In the past two years I have: 1) purified the two mammalian cobalamin (vitamin B12)-dependent enzymes, methylmalonyl-CoA mutase and N5-methyltetrahydrofolate homocysteine methyltransferase, to homogeneity from human placenta; 2) synthesized a variety of cobalamin analogues and determined that they can be delivered to many tissues in large amounts; and 3) found that a number of previously unrecognized natural cobalamin analogues are present in human as well as mammalian sera and tissues. I propose to use the purified cobalamin-dependent enzymes, the synthetic cobalamin analogues and the natural cobalamin analogues, to elucidate the metabolic role of cobalamin in mammalian cells. To purified cobalamin-dependent enzymes will be utilized to develop a radioimmunoassay to study the regulation of enzyme protein in normal cells and in cells from patients with a variety of genetic defects in the synthesis of coenzyme cobalamin and in the synthesis of the apo-enzymes. The results of these studies could have important therapeutic implications for these genetic disorders. The recently discovered natural cobalamin analogues will be studied to determine if they play a role in the development of the hematologic and/or neurologic manifestations of cobalamin deficiency in humans and if they play a role in other neurologic diseases such as multiple sclerosis. I will also determine if natural and synthetic cobalamin analogues can be utilized to develop an animal model of these hematologic and neurologic abnormalities and thus elucidate their biochemical basis. Finally, the natural and synthetic analogues will be tested for their ability to preferentially inhibit malignant cell proliferation in tissue culture and in tumor bearing animals. Results of these studies could lead to a new class of chemotherapeutic agents for human malignancies.